In 2009, several projects as outlined in the goals and objectives, gained momentum as a result of hiring three post-doctoral fellows and one post-baccalaureate student. Among the projects towards which we made progress were;T cell receptor (TCR) conformational analysis, signaling via low affinity TCR ligands, imaging the dynamics of the Fos transcription factor in T cells. One of our goals is to develop spectroscopic techniques to understand how multiple subunit containing receptors such as antigen receptors whose ligand binding subunit is different from the signal transducing subunit, communicate information from ligand binding to signal transduction. Fluorescence Resonance Energy Transfer (FRET) is a fluorescence phenomenon in which fluorophores transfer energy among themselves only when they are in molecular proximity of each other. Hence if FRET measurements are done at high time resolution, it offers the possibility of studying the dynamics of individual subunits within a receptor complex. Fluorescence Correlation Spectroscopy (FCS) is a technique that allows the measurement of diffusion coefficients of fluorophores by statistical analysis of fluctuations in fluorescence intensities measured from a confocal volume. These measurements are done using a single photon detector known as an avalanche photodiode and offer very high time resolution of the order of nano-seconds. Hence combining FRET and FCS will allow us to monitor FRET events at the time scales of subunit dynamics. This technique also has the potential of measuring molecular conformations within a single protein molecule. We are in the process of building a custom microscope which will allow us to make these measurements. We will use this technique to address two questions. Are there molecular motions in the ligand binding TCR-alpha-beta subunit that we can measure upon receptor engagement? What are the dynamics of subunits of the cytokine receptors belonging to the common gamma chain family? In collaboration with David Margulies, we have received help in designing positions of KKCK (lysine-lysine-cysteine-lysine) motifs in the extracellular domains of alpha and beta chains of TCR. KKCK motif has a high affinity for a maleimide labeling reaction compared to an isolated cysteine offering the possibility of site specific labeling of TCR on the cell surface. We have engineered a green fluorescent protein (GFP) as a tag on the cytoplasmic side of the AND-TCR beta chain and we will site specifically attached an Alexa546 dye on the engineered sites in the extracelluar domain. GFP and Alexa546 form an excellent FRET pair and hence will be best suited for these measurements. We have generated these mutant constructs and verified that they assemble with the CD3 chains and express on the surface of TCR beta chain deficient Jurkat Cell line. We have also found conditions under which we can label these receptors at the cell surface. We still need to determine the specificity of the labeling reaction. We will perform two kinds of measurements with these reagents. First using TIRF microscopy we will determine if the extent of FRET in these engineered receptors are different in engaged vs unengaged receptors by comparing measurements in and outside of microclusters. Second, we will perform FRET-FCS experiments on purified labeled receptors to get an insight into the molecular motions in the receptors. To address the second question regarding communication between subunits we have made constructs tagging the Cyan Fluorescent Protein (CFP) and Yellow Fluorescent Protein (YFP) to the cytoplasmic side of individual subunits of common gamma chain cytokine receptors. Since CFP and YFP form a good FRET pair we will be able to perform FRET-FCS measurements and understand how subunits interact with each other. We have a deep interest in understanding the signaling pathways activated downstream of TCR in response to low affinity ligands. Our hypothesis based on previously published report is that low affinity ligands activate the Ras pathway via recruitment of Grb2-Sos module to the TCR complex in the absence of LAT phoshorylation and recruitment. We will transfect AND T cells with GFP tagged LAT, Zap-70, Grb2 and Sos and visualize their recruitment to sites of antigen engagement in TCR microclusters using TIRF microscopy to test this hypothesis. We have generated all these GFP fusion constructs and have optimized conditions in which we can transfect AND T cells using Amaxa based electroporation. We will visualize antigen engagement by imaging fluorescent antigenic peptides loaded on to soluble histidine tagged Ek molecules. The fluorescent peptides have been generated in house using FMOC chemistry ensuring a single fluorophore per peptide molecule. Our goal is to finally move into addressing questions of differentiation of T cells during influenza infection. To this end we have obtained three transgenic mice that express TCRs specific for the influenza protein hemagglutinin (HA). These TCRs are E-d and A-d restricted. To perform imaging experiments we have generated constructs to express his-tagged soluble versions of E-d and A-d in insect cells so that we can load them with HA derived peptides and incorporate them in glass supported lipid bilayers. One of our key goals is to understand the relationship between TCR engagement events at the cell surface and activation of transcription factors in the nucleus. For these experiments we have obtained transgenic mice that express a fusion protein between the transcription factor Fos and GFP under the control of the Fos promoter. Fos is not expressed in T cells in the basal state and is induced upon signaling. We have crossed these mice to AND TCR transgenic mice and have begun to study the induction of Fos using single cell imaging in response to ligands of varying strengths. We find that Fos is induced within 20 minutes of interaction with antigen and continues to accumulate for at least 2 more hours. Given that it takes about 6-7 minutes for GFP to mature, it is likely that the kinetics of Fos induction are much faster. We also find that Fos fails to get induced in response to super agonist signaling, a result that was unexpected. We have also generated constructs of NFAT and p65 fused to tag-RFP (a red fluorescent protein) which we are simultaneously expressing in these cells so that we can follow the dynamics of two transcription factors at the same time. We are using endogenous promoters to express these transcription factors in T cells so that they are expressed at physiological levels and we are learning a lot regarding the requirements of expression of p65 in T cells. These reagents will allow us to address multiple questions concerning the single cell dynamics of transcription factors which cannot be addressed using biochemical analysis. In an ongoing collaboration with Pam Schwartzbergs lab we are investigating the role of the adapter protein SAP in TCR signaling and immunological synapse formation. The SAP molecule is associated with X-linked lympho-proliferative diseases and interacts with the cytoplasmic tails of SLAM family member proteins. We find that activated SAP deficient AND T cells form normal synapses. We have purified soluble his-tagged and GPI-anchored forms of SLAM family members LY108 and CD84. These proteins will be incorporated in glass supported lipid bilayers to study the impact of synapse formation by SAP-deficient T cells. We will also perform these experiments under shear flow as SAP-deficient T cells exhibit defective conjugate persistence in-vivo.